We want to understand the molecular mechanisms that underlie antibody somatic hypermutation (SHM) and class switch DNA recombination (CSR) in systemic autoimmunity, particularly systemic lupus. Like antibodies to microbial pathogens, most pathogenic autoantibodies are somatically hypermutated and class-switched. SHM introduces mainly point-mutations in immunoglobulin (Ig) heavy and light chain V(D)J regions, thereby providing the substrate for selection by (self) antigen of higher affinity (auto)antibody mutants. CSR replaces the expressed IgH constant (CH) region, e.g., C5, with downstream C3, C1 or C5, thereby endowing (auto)antibodies with new biological effector functions. Both SHM and CSR entail two sequential stages: (i) generation of DNA lesions, as mediated by activation-induced cytidine-deaminase (AID), and (ii) DNA lesion repair by the basic site bypass, base-excision repair (BER) or mismatch repair (MMR) machineries, leading to the emergence of mismatches (SHM) or double-strand DNA breaks (DSBs) and their resolution (CSR). We argue here that in systemic lupus, SHM and CSR are dysregulated, as a result of upregulated AID and, therefore, increased DNA lesions, and altered DNA repair. We contend that the phylogenetically conserved homeodomain protein HoxC4, which, as we have shown, critically regulates SHM and CSR, induces AID expression by directly binding to a conserved HoxC4/Oct-binding 5'-ATTTGAAT-3' site we have identified in the AID promoter. We also contend that estrogen greatly enhances CD154:CD40-induced HoxC4 expression by triggering the binding of estrogen receptors (ERs) to two conserved estrogen responsive elements (EREs) we have recently identified in the HoxC4 promoter, thereby further enhancing AID expression and contributing to SHM/CSR dysregulation in lupus. We also argue that the translesion DNA synthesis (TLS) polymerase (pol) 8 and pol7 are critical in SHM of (auto)antibodies, by repairing DNA lesions after being recruited by ubiquitinated PCNA, which mediates the polymerase switch from high-fidelity replicative DNA polymerases to error-prone TLS polymerases. Finally, we hypothesize that expression of HoxC4, AID and TLS polymerases is dysregulated in lupus-prone mice and ablation of HoxC4, Ung or TLS pol7 leads to decreased SHM/CSR and, therefore, decreased level of high-affinity and isotype-switched autoantibodies and delayed immunopathology. To test our hypotheses, we will: (i) define the molecular mechanisms of HoxC4-mediated AID promoter activation and the enhancement of HoxC4 and, therefore, AID expression by estrogen; (ii) address the role of TLS pol8 and pol7 and their recruitment by ubiquitinated PCNA in SHM; and, finally, (iii) analyze the SHM/CSR dysregulation and the impact of deficiency of HoxC4, Ung org pol7 on the generation of hypermutated and class-switched autoantibodies in lupus-prone mice. These experiments will use sophisticated biochemical methods and our new KO HoxC4-/-, pol8-/-, double KO pol8-/-Ung-/- and pol7-/-Ung-/- and mutant Aicdatm1(Pmut) and pol7mut mice, and lupus-prone MRL/Faslpr/lpr mice deficient in HoxC4, Ung or pol7. PUBLIC HEALTH RELEVANCE: This proposal addresses the molecular mechanisms of the generation of pathogenic autoantibodies in systemic lupus erythematosus (SLE): immunoglobulin (antibody) somatic hypermutation (SHM) and class switch DNA recombination (CSR). By increasing the affinity of antibodies for antigens and by changing the constant region of the antibody from IgM to IgG, IgA and/or IgE, SHM and CSR play a central role in the generation of highly specific and advantageous antibody responses to microbial pathogens and tumors. However, by increasing the affinity and changing the class, mainly to IgG, of antibodies to components of the body, SHM and CSR can also lead to the generation of autoantibodies that can damage tissues and organs (pathogenic autoantibodies). Thus, dysregulation of SHM and CSR can lead to the emergence of autoimmunity, including SLE. By addressing the mechanisms underlying SHM and CSR and their dysregulation, these studies will help understand how specific pathogenic autoantibodies, as those occurring in patients with SLE, are generated in the human body, eventually leading to the development of better therapeutic approaches.